Hematopoietic stem cell proliferating agents

ABSTRACT

This invention relates to a hematopoietic stem cell proliferating agent comprising IGF-I, a hematopoietic stem cell proliferating agent comprising IGF-I and at least one protein selected from among SCF, M-CSF, and G-CSF, and a method of growing hematopoietic stem cells which comprises culturing hematopoietic stem cells in a medium containing IGF-I and at least one protein selected from the group consisting of SCF and M-CSF.  
     The hematopoietic stem cell proliferating agent of the invention causes hematopoietic stem cells to proliferate in the undifferentiated state whether in vivo or in vitro and can, therefore, be used for amelioration of the cytopenia induced by radiotherapy or chemotherapy using anticancer drugs, prevention of infectious diseases associated with lymphopenia, or in vitro culture for multiplication of hematopoietic stem cells and extrasomatic culture of recombinant stem cells in gene therapy.

TECHNICAL FIELD

[0001] This invention relates to a hematopoietic stem cell proliferatingagent and a method for stimulating the proliferation. More particularly,the invention relates to a hematopoietic stem cell proliferating agentcomprising insulin-like growth factor I either alone or in combinationwith some or other colony-stimulating factors and/or growth factors andto a method for proliferating.

BACKGROUND ART

[0002] A variety of growth factors relating to the blood system areunder investigation, and erythropoietin (EPO) as an erythropoiesisstimulating agent and granulocyte colony-stimulating factor (G-CSF) as aleukopoiesis stimulating agent, among others, have been clinically putto use to this day. Regarding the technology for proliferating ofhematopoietic stem cells, various cytokines such as stem cell factor(SCF), macrophage colony-stimulating factor (M-CSF), etc. were exploredbut none has been found to be effective enough in causing hematopoieticstem cells to multiply sufficiently in the undifferentiated form.

[0003] As the result of an intensive investigation, the inventors ofthis invention found that hematopoietic stem cells can be successfullycaused to multiply in the undifferentiated state well by usinginsulin-like growth factor I (IGF-I) in combination with at least oneprotein selected from among SCF, M-CSF, and G-CSF. This invention hasbeen developed on the basis of the above finding.

DISCLOSURE OF THE INVENTION

[0004] This invention relates to a hematopoietic stem cell proliferatingagent comprising IGF-I, a hematopoietic stem cell proliferating agentcomprising IGF-I and at least one protein selected from among SCF,M-CSF, and G-CSF, and a method of stimulating proliferation ofhematopoietic stem cells which comprises culturing hematopoietic stemcells in a culture medium containing IGF-I and at least one proteinselected from among SCF, M-CSF, and G-CSF. Furthermore, this inventionrelates to a method of proliferating of hematopoietic stem cells in amammal which comprises using IGF-I alone or in combination with at leastone protein selected from among SCF, M-CSF, and G-CSF.

[0005] Since the hematopoietic stem cell proliferating agent and methodof the invention are effective in causing hematopoietic stem cells tomultiply in the undifferentiated state whether in vivo or in vitro, theinvention finds application in the management of the cytopenia inducedby radiotherapy or chemotherapy using anticancer drugs, prevention ofinfectious diseases with which lymphopenia is associated, treatment ofmyelopathy inclusive of osteomyelodysplasia and bone marrow suppression,therapy of marrow diseases such as leukemia-advanced renalimpairment-bone marrow suppression, improvement in engraftment survivalin bone marrow transplantation, therapy of hypocytosis associated withinherited diseases, in vitro culture for multiplication of hematopoieticstem cells, and extrasomatic culture of recombinant stem cells in genetherapy, among other uses.

[0006] The IGF-I which can be used in this invention includes thecorresponding proteins derived from human, bovine, and other mammals byrecombinant DNA technology (e.g. Kokai Tokkyo Koho S61-1396 for IGF-I),peptide synthesis, cell culture, or other technology and even themuteins having IGF-I activity which can be derived from the recombinantor other IGF-I by partial modification of its amino acid sequence bysubstitution, insertion, addition, or deletion of one or more amino acidresidues (e.g. WO89/05822).

[0007] The SCF, M-CSF, or G-CSF which can be used in this inventionincludes the corresponding proteins derived from human, bovine, or othermammals by recombinant DNA technology, peptide synthesis, cell culture,or other technology and even those muteins having SCF, M-CSF, or G-CSFactivity which can be derived from the recombinant or other SCF, M-CSFor G-CSF by partial modification of its amino acid sequence bysubstitution, insertion, addition, or deletion of one or more amino acidresidues. Here, the sugar chain may be present or absent.

[0008] The hematopoietic stem cell proliferating agent of this inventionwhich comprises IGF-I and either SCF, M-CSF, or G-CSF can beadministered virtually without limitations on the dosage form or forms,sequence of administration, or route of administration, all that isnecessary being to insure that those factors will be concurrentlyavailable in the recipient's body. For example, they can be administeredas a mixture in a single dosage form or in independent dosage forms,whether concurrently or one after another, and whether by the same routeor by different routes.

[0009] The hematopoietic stem cell proliferating agent of the inventionis generally provided in an oral dosage form or in a nonoral dosageform, e.g. an injection, drip infusion, transdermal therapeutic system,transnasal therapeutic system, external preparation, suppository, etc.,each containing IGF-I alone or IGF-I plus at least one protein selectedfrom among SCF, M-CSF and G-CSF together with a carrier (e.g. distilledwater for injection, physiological saline, glucose injection, etc.), astabilizer (e.g. albumin, sodium citrate, arginine, dextran, etc.), a pHcontrol agent (e.g. sodium monohydrogen phosphate, sodium dihydrogenphosphate, etc.) and other additives. Such dosage forms or systems mayfurther contain one or more growth factors such as SCF, M-CSF, G-CSF,EPO, and IL-3.

[0010] The dosage of this hematopoietic stem cell proliferating agentdepends on the patient's body weight, sex, and clinical condition butthe dose level for an adult human is generally about 1˜1000 μg/kg interms of IGF-I and preferably about 5˜500 μg/kg on the same basis. WhenSCF, M-CSF, or G-CSF is used concomitantly, SCF, M-CSF or G-CSF can beformulated in the same amount as IGF-I or in an amount ranging from 0.01to 100 times the amount of IGF-I.

[0011] This hematopoietic stem cell proliferating agent can beadministered orally or otherwise, e.g. by intravenous injection,intravenous drip, subcutaneous injection, coronary intraarterialadministration, transdermal administration, transnasal administration,or rectal administration.

[0012] IGF-I and any of SCF, M-CSF and G-CSF can be formulated in oneand the same dosage form but may be administered independently one afterthe other or concurrently, either by the same route or different routes.When they are administered in sequence, it does not matter which isadministered first.

[0013] When the hematopoietic stem cell proliferating agent of theinvention is used for ameliorating the cytopenia induced by radiotherapyor chemotherapy using anticancer drugs, the hematopoietic stem cellproliferating agent of the invention can be used alone or concurrentlywith EPO, M-CSF, SCF, IL-3, G-CSF, and/or the like. When it is used forthe prevention of infectious diseases associated with lymphopenia,GM-CSF or the like can be used concomitantly. Furthermore, in thetreatment of myelopathies such as osteomyelodysplasia and bone marrowsuppression or marrow diseases such as leukemia-advanced kidneyimpairment-bone marrow suppression, for improvement in engraftmentsurvival in bone marrow transplantation, or in the treatment ofhypocytosis associated with inherited diseases, suitable growth factorssuch as EPO, G-CSF, GM-CSF, etc. can be used as concomitant medications.

[0014] In the in vitro culture for multiplication of hematopoietic stemcells or extrasomatic culture of recombinant stem cells in gene therapy,among other applications, the hematopoietic stem cell proliferatingagent of the invention can be used in combination with other suitablegrowth factors. In vitro culture of marrow cells can be essentiallycarried out in accordance with the method described in Shin SeikagakuJikken Koza [New Biochemical Experiment Series] 18 Saibo Baiyo Gijutsu[Cell Culture Technology] (ed. by Japanese Biochemical Society, TokyoKagaku Dojin, 1989). For example, using a CO₂ incubator, marrow cellscan be cultured in RD medium [RPMI1640:DMEM=1:1 (v/v)] supplemented withinsulin, transferrin, 2-mercaptoethanol, ethanolamine, selenious acid,HEPES, etc. in the presence of IGF-I (1˜1000 μg/ml) and at least oneprotein selected from among SCF (1˜1000 μg/ml) M-CSF (1˜1000 μg/ml), andG-CSF (1˜1000 μg/ml).

BEST MODE FOR CARRYING OUT THE INVENTION

[0015] This invention is now described in further detail by way of thefollowing examples.

EXAMPLE 1 Procurement of Hematopoietic Stem Cells

[0016] Using the femora of male C57BL mice (10 individuals), marrowcells were recovered in α-MEM (5 ml, Nikken Biomedical ResearchInstitute). This cell suspension was centrifuged (1,200 rpm, 10 min.),the supernatant was aspirated off, and the cells were resuspended in 10%FCS-α-MEM (5 ml). By this procedure, approximately 3×10⁷ marrow cellsper mouse were harvested.

EXAMPLE 2 FACS

[0017] From the marrow cells thus procured, Sca-1⁺, Lin⁻, andc-kit⁺cells were collected using Fac-Scan flow cytometer(Becton-Dickinson). The labeled antibodies used are shown in Table 1. Itis known from the literature that the cell fraction thus obtainedcontains murine hematopoietic stem cells [Okada, S. et al. (1992), Blood80, 3044-3050]. TABLE 1 Labeled antibodies used in FACS Marker Labeledantibody Specificity Lin Biotinylated anti-CD3 ε (Clone T cell 500A2)Biotinylated anti-CD45R (B220) B cell Biotinylated anti-mouseErythrocyte erythrocytes (TER119) Biotinylated anti-CD11b (Mac-1)Monocyte/ macrophage Biotinylated anti-myelocyte Granulocytedifferentiation antigen (Gr1) Sca-1 PE-labeled anti-mouse SCA-1 (E13-Stem cell 161.7) c-kit PITC-labeled anti-mouse CD117 (3CI) SCF receptor

EXAMPLE 3 Action of IGF-I

[0018] A U-bottomed 96-well microtiter plate (Nunc, Denmark) was seededwith hematopoietic stem cells at a density of 50 cells/10% FCS-α-MEM(100 μg 1)/well.

[0019] To each well was added IGF-I (Mecasermin, recombinant, FujisawaPharmaceutical) (100 ng/ml) SCF (recombinant, Genzyme) (1.5, 3.0, 6.0,12.5, 25 ng/ml)+IGF-I (100 ng/ml), or M-CSF (recombinant, R&D System)(0.1, 0.3, 1.0, 3.0, 10, 30, 100 ng/ml)+IGF-I (100 ng/ml) and the platewas incubated in a CO₂ incubator at 37° for 10 days.

[0020] After 6 days of culture, the cells in each well were counted.

[0021] After 10 days of culture, the medium was aspirated off and thecytoplasmic acid phosphatase activity was assayed by the methoddescribed in the literature [Ueda et al. (1994), Neurosci. Lett., 165,203-207].

EXAMPLE 4 Results

[0022] The results are summarized in Table 2. Table 2 Effect of thecombination of IGF-I with either SCF or M-CSF IGF-I (ng/ml) Factor added(ng/ml) Δ 450 means ± SD 100 SCF 25.0 1.387 ± 0.207 100 SCF 12.5 0.654 ±0.273 100 SCF 6.0 0.056 ± 0.015 100 SCF 3.0 0.035 ± 0.010 0 SCF 25.00.176 ± 0.074 100 M-CSF 100.0 0.906 ± 0.645 100 M-CSF 30.0 0.778 ± 0.649100 M-CSF 10.0 0.564 ± 0.402 100 M-CSF 3.0 0.064 ± 0.050 0 M-CSF 100.00.052 ± 0.045 100 None ND

[0023] It will be apparent from Table 2 that IGF-I as used incombination with SCF or M-CSF increased the cytoplasmic acid phosphataseactivity of the marrow cell fraction obtained in Example 2. It is knownthat cytoplasmic acid phosphatase activity increases in proportion to anincrease in the cell population. Therefore, it is clear that IGF-I asused in combination with SCF or M-CSF exerted a hematopoietic stem cellproliferation stimulating action. It is known from the literatrue thatSCF and M-CSF each independently does not show hematopoietic stem cellproliferation stimulating activity [Okada, S. et al. (1992), Blood 80,3044-3050] and the inventors verified the finding.

EXAMPLE 5 FACS

[0024] Using the same procedure as used in Example 2, Lin⁻, Sca-1⁺,c-kit⁺, and CD34⁻ cells were harvested. For selection of CD34-cells,biotinylated anti-mouse CD34 (RAM34) (Fermigen, San Diego, Calif.) wasnewly used. Those cells accounted for about 0.04% of the total marrowcell population. It is known from the literature that cells of thisgroup are hematopoietic stem cells (Ohsawa & Nakauchi 1995, JapaneseMolecular Biochemical Society S4B-3; Ohsawa et al. 1995, Synopsis ofBlood Stem Cell Symposium).

EXAMPLE 6 Effect of IGF-I

[0025] Using ACDU, the cells obtained in Example 5 were transferred to96-well plates, one cell per well, and about 50 wells were used as onegroup.

[0026] To each well was added SCF (25 ng/ml)+IGF-I (100 ng/ml) , M-CSF(100 ng/ml)+IGF-I (100 ng/ml), or SCF (25 ng/ml)+IL-3 (10 ng/ml), andthe plates were incubated in a CO₂ incubator at 37° C. for 10 days.

[0027] After 6 days of culture, the cells in each well were counted.

[0028] After 10 days of culture, the medium was aspirated off from eachwell and the cytoplasmic acid phosphatase activity was assayed.

EXAMPLE 7 Results

[0029] The results are summarized in Table 3. Table 3 Effect of thecombination of IGF-I with either SCF or M-CSF on the single cell Meannumber of Surviving wells/ surviving all wells cells/ (survival rate)well IGF-I (100 ng) + 25/47 (53%) 10 SCF (25 ng/ml) IGF-I (100 ng) + 3/47 (6.0%)  2 M-CSF (100 ng/ml) Medium  0/23 (0.0%)  0 IL-3 (10 mg) +33/48 (69%) NT SCF (25 ng/ml)

[0030] It will be apparent from Table 3 that IGF-I as used incombination with SCF or M-CSF is capable of causing proliferation ofsingular hematopoietic stem cells.

EXAMPLE 8 Immunostaining

[0031] Sca-1⁺, Lin⁻, and c-kit⁺ cells were collected by the sameprocedure as in Examples 1 and 2. The harvested cells were seeded on a100-well chamber slide (Lab-Tek, Nunc). In a CO₂ incubator at 37° C.,the cells were cultured in 10% FCS-α-MEM (100 μl) containing growthfactors [IGF-I (100 ng/ml) and SCF (25 ng/ml)] for 8 days.

[0032] The cultured cells were fixed with acetone (−20° C.) on theslide. After addition of 1% BSA-phosphate buffer for inhibition ofnonspecific binding, the cells were treated with the antibodies shown inTable 4.

[0033] The cells were not stained by any of the antibodies used,indicating that they had no tendency toward differentiation to anyspecific kinds of offspring cells. The augmentation of cell populationby IGF-I plus SCF was not the proliferation due to differentiation ofstem cells but the proliferation of undifferentiated stem cells. TABLE 4Labeled antibodies used in FACS Marker Labeled antibody Specificity LinBiotinylated anti-CD3 ε (Clone T cell 500A2) Biotinylated anti-CD45R(B220) B cell Biotinylated anti-mouse Erythrocyte erythrocytes (TER119)Biotinylated anti-CD11b (Mac-1) Monocyte/ macrophage Biotinylatedanti-myelocyte Granulocyte differentiation antigen (Gr1) Sca-1PE-labeled anti-mouse SCA-1 (E13- Stem cell 161.7)

EXAMPLE 9 The Action of Human G-CSF Plus Human IGF-I

[0034] Hematopoietic stem cells were transferred to a U-bottomed 96-wellmicrotiter plate (Nunc, Denmark), 50 cells/10% FCS-α-MEM (100 μl)/well.

[0035] To each well was added human IGF-I (Mecasermin, recombinant,Fujisawa Pharmaceutical) (100 ng/ml) or human G-CSF (recombinant, R&DSystem) (5, 50, or 500 mg/ml)+IGF-I (100 ng/ml), and the plate wasincubated in a CO₂ incubator at 37° C.

[0036] After 7 days of culture, the medium was aspirated off from eachwell and the cytoplasmic acid phophatase activity was assayed by themethod described in the literature (Ueda et al. (1994), Neurosci. Lett.,165, 203-207).

EXAMPLE 10 Results

[0037] The results are summarized in Table 5. TABLE 5 G-CSF (ng/ml)IGF-I (ng/ml) Δ 450 mean ± SE 500 100 0.164 ± 0.033 50 100 0.132 ± 0.0865.0 100 0.126 ± 0.071 0.50 100 0.038 ± 0.020 0 100 0.001 ± 0.004 500 00.054 ± 0.007 50 0 0.015 ± 0.019 5.0 0 0.010 ± 0.014 0.50 0 0.008 ±0.003 0 0 0.000 ± 0.002

[0038] It will be apparent from Table 5 that IGF-I as used incombination with G-CSF enhanced the cytoplasmic acid phosphataseactivity of the marrow cell fraction obtained in Example 2. It is knownthat cytoplasmic acid phosphatase activity is positively correlated withthe number of cells. Therefore, it is evident that IGF-I as used incombination with G-CSF exhibited a hematopoietic stem cell proliferationstimulating action. No effect was found with G-CSF alone.

EXAMPLE 11 The Action of Human SCF Plus Human IGF-I

[0039] Hematopoietic stem cells were transferred to a U-bottomed 96-wellmicrotiter plate (Nunc, Denmark), 50 cells/10% FCS-α-MEM (100 μl)/well.

[0040] To each well was added human SCF (recombinant, R&D System) (5,50, or 500 mg/ml)+IGF-I (100 ng/ml) and the plate was incubated in a CO₂incubator at 37° C.

[0041] After 7 days of culture, the cells in each well were counted.

EXAMPLE 12 Results

[0042] The results are summarized in Table 6. TABLE 6 Mean ± SE Mean ±SE SCF (ng/ml) (IGF-I 100 ng/ml) (IGF-I 0 ng/ml) 800  200 ± 0.0  75.7 ±26.4 400  175 ± 21.5 36.0 ± 14.3 200 58.0 ± 16.7 14.8 ± 8.2  100 31.5 ±6.2  9.8 ± 1.6 50 16.0 ± 5.5  6.5 ± 5.0 25 6.3 ± 2.9 3.5 ± 0.5 12.5 3.8± 1.5 2.0 ± 0.7 6.25 3.3 ± 0.8 3.0 ± 3.7 3.0 3.5 ± 1.1 2.3 ± 1.1 0 2.8 ±2.2 1.0 ± 1.0

[0043] It will be apparent from Table 6 that IGF-I as used incombination with SCF stimulated proliferation of hematopoietic stemcells to a remarkable extent. This combination is, therefore, useful forthe maintenance and multiplication of hematopoietic stem cells.

EXAMPLE 13 Pharmaceutical Preparations

[0044] The components indicated in Table 7 were dissolved in water andvials filled with 5 ml portions of the respective solutions werelyophilized to provide hematopoietic stem cell proliferating agents.TABLE 7 Examples of the hematopoietic stem cell proliferationstimulating composition Human IGF-I SCF M-CSF G-CSF albumin (mg) (mg)(mg) (mg) (mg) Composition 1 10 0 0 0 0 Composition 2 10 0 0 0 50Composition 3 10 20 0 0 50 Composition 4 10 0 10 0 50 Composition 5 10 00 10 50 Composition 6 5 10 10 0 0 Composition 7 5 0 5 5 100 Composition8 5 10 0 5 100 Composition 9 5 5 5 5 50

INDUSTRIAL APPLICABILITY

[0045] Thus, the hematopoietic stem cell proliferating agent and methodof the invention are of great utility value, for hematopoietic stemcells can be caused to proliferate in the undifferentiated state whetherin vivo or in vitro.

1. A hematopoietic stem cell proliferating agent comprising IGF-I.
 2. Ahematopoietic stem cell proliferating agent according to claim 1 whichcomprises IGF-I and at least one protein selected from the groupconsisting of M-CSF, SCF, and G-CSF.
 3. The hematopoietic stem cellproliferating agent according to claim 2 which comprises M-CSF andIGF-I.
 4. The hematopoietic stem cell proliferating agent according toclaim 2 which comprises SCF and IGF-I.
 5. The hematopoietic stem cellproliferating agent according to claim 2 which comprises G-CSF andIGF-I.
 6. A method of growing hematopoietic stem cells which comprisesculturing hematopoietic stem cells in a medium containing IGF-I and atleast one protein selected from the group consisting of M-CSF, SCF, andG-CSF.
 7. A method of growing hematopoietic stem cells which comprisesadministering IGF-I to a mammal.
 8. The method of growing hematopoieticstem cells according to claim 7 which comprises administering IGF-I andat least one protein selected from the group consisting of M-CSF, SCF,and G-CSF.
 9. The method of growing hematopoietic stem cells accordingto claim 8 which comprises administering IGF-I and M-CSF.
 10. The methodof growing hematopoietic stem cells according to claim 8 which comprisesadministering IGF-I and SCF.
 11. The method of growing hematopoieticstem cells according to claim 8 which comprises administering IGF-I andG-CSF.
 12. Use of IGF-I for stimulating proliferation of mammalianhematopoietic stem cells.
 13. Use of IGF-I and at least one proteinselected from the group consisting of M-CSF, SCF, and G-CSF forstimulating proliferation of mammalian hematopoietic stem cells.
 14. Useof IGF-I and M-CSF for stimulating proliferation of mammalianhematopoietic stem cells.
 15. Use of IGF-I and SCF for stimulatingproliferation of mammalian hematopoietic stem cells.
 16. Use of IGF-Iand G-CSF for stimulating proliferation of mammalian hematopoietic stemcells.